MHC class II-restricted antigen processing depends upon a complex series of events involving: a) the assembly of class II alphabeta-invariant chain nonamers in the endoplasmic reticulum (ER); b) their transport through the Golgi apparatus and delivery into the endosomal/lysosomal system; c) partial disassembly of the complexes by invariant chain proteolysis; d) loading of peptides derived from internalized proteins into released alphabeta dimers; e) delivery of the peptide-loaded alphabeta dimers tot he plasma membrane. This proposal seeks to further define the role in assembly of calnexin and other chaperones and to examine the role of calnexin and the p35 and p43 forms of the invariant chain in ER retention of alphabeta-invariant chain complexes. It also seeks to determine whether the p41/p43 forms of the invariant chain play a particular role in assembly by virtue of their ability to interact with free class II alpha- and beta- subunits and/or whether this property is important in the endosomal disassembly of alphabeta-invariant chain complexes and the peptide loading process. The effects of coexpression with p43/p41 versus p35/p33 on the spectrum of peptides associated with class II molecules will also be compared. To further understanding of class II processing mechanisms in professional antigen presenting cells, studies will be initiated of class II-positive human macrophage lines, one, Mono Mac 6, which is naturally class II positive, and others which will be rendered class II-positive by transfection with the class II transactivating gene (CIITA). Class II assembly and transport, the interaction of class Ii molecules with the phagocytic and endocytic systems of the macrophage, alphabetaI disassembly and peptide loading, and antigen presentation will be examined in these model systems. The HLA-DM genes involved in antigen processing were identified by change isolation of mutants in cells hemizygous for the HLA complex, which facilitated their identification. Important genes unlinked to the MHC are difficult to identify by mutagenesis because of homozygosity and the requirement for double mutants. CIITA induces the expression of all class II structural genes, the invariant chain gene and the HLA-DM genes. It is hypothesized that other genes important for class II- restricted antigen processing are likely to be co-regulated by CIITA, and it is proposed to isolate and identify such genes by subractive hybridization using cDNA from class II-negative cells and transfectants of these cells expressing CIITA. Overall, this proposal is designed to further our understanding of the critical intracellular mechanisms involved in antigen processing.